This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To define the influence of endometrial cells on the mibration of trophoblasts differentiated from human embryonic stem cells. In the placenta, the differentiation of cytotrophoblasts (CTB) to the migratory extravillous trophoblasts (EVTB) is determined by many factors including the surrounding extracellular matrix, integrin signaling, and the immune cells present in the maternal decidua. Trophoblasts have been differentiated from human embryonic stem cells (hESC) in vitro by EB formation and by treatment with BMP4. We therefore sought to test whether there was an effect of secreted factors from human umbilical vein endothelial cells (HUVECs) on EB-derived trophoblasts migration. Additionally, we were interested to determine whether specific chemokines secreted by natural killer (NK) cells, an abundant immune cell in the human decidua, would also effect EB-derived trophoblast migration. Briefly, we formed EBs of uniform size (1000-cells) and shape via forced aggregation. We then seeded 50 EB/invasion insert and either placed the insert in a well containing a monolayer of HUVECs or wells with varying concentrations of recombinant chemokines IL-8, IP-10, and MCP-1 (20, 100, 500 ng/ml). We allowed the EB-derived trophoblasts to migrate for 5 days and migrated cells were stained with calcein-AM and quantified using the NIH ImageJ 1.32j software. Our results indicate that EB-derived trophoblast migration continues for up to 96 hours in invasion chambers. In addition, HUVECs have a positive influence on EB-derived trophoblast migration and additionally were found to secrete IL-8 and MCP-1 in culture. IL-8, IP-10, and MCP-1 are also secreted by NK cells in the maternal decidua. Finally, the presence of IL-8 at 100 ng/ml significantly increased migration. Overall, these experiments effectively show the effects of chemokines secreted by maternal endothelial cells and NK cells on EB-derived trophoblast migration. This further establishes the EB-derived trophoblast model as a useful tool to study EVTB differentiation and migration during placental development. This work used WNPRC Stem Cell Resources.